Tobacco specific nitrosamines (TSNA) are generally considered to be undesirable constituents that occur naturally in cured or dried leaves of tobacco. Tobacco specific nitrosamines, including N′-nitrosonomicotine (NNN), N′-nitrosoanatabine (NAT), N′-nitrosoanabasine (NAB), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are the direct result of a chemical reaction between certain tobacco alkaloids that are endogenous to tobacco and unstable NOx radicals, such as nitrite (NO2), that are formed readily in tobacco during the curing process (Cui M., Yang, H., Bush, L. P. and Burton, H., Tob. Sci. Res. Conf. 50, Abstr. 74, 1996). It is generally understood that microbes on or in the tobacco plant before, during, and after curing are most responsible for the formation of nitrite (NO2), the predominant NOx precursor for TSNA formation (Bush L. P., M. Cui, H. R. Burton, F. F. Fannin, L. Lei, and N. Dye, Recent Advances in Tobacco Science, 27, 23-46, 2001). Reducing the microbial population during tobacco curing may limit substrate (NO2) availability and result in lower levels of TSNAs.
Tobacco harvested from the field is cured using a variety of practices that may include natural air-curing, forced, heated air-curing known as flue-curing, and fire-curing, a process in which wood or wood by-products such as sawdust are ignited to produce heat and smoke within the curing structure (Tso T. C., Production, physiology and biochemistry of tobacco plant, IDEALS Inc. Beltsville, Md., 1990.; Davis D. L and M. T. Nielsen, Tobacco production, chemistry and technology (World Agricultural Series, 1999 CORESTA, ISBN-0-632-04791-7), 1999). Tobacco curing is a process of physical and biochemical changes that bring out the aroma and flavor of each variety of tobacco. The physical changes are witnessed by moisture reduction and color change. The biochemical changes are witnessed by the degradation of chlorophyll that brings leaves their yellow appearance and the converting starch into sugar (Tso T. C., Production, physiology and biochemistry of tobacco plant, IDEALS Inc. Beltsville, Md., 1990.; Davis D. L. and M. T. Nielsen, Tobacco production, chemistry and technology (World Agricultural Series, 1999 CORESTA, ISBN-0-632-04791-7), 1999). Curing involves three essential steps: yellowing, browning (leaf drying) and stem drying. The yellowing stage is a continuation of the ripening process and is thought to be the most important part of the curing process. The leaf is still biochemically active till the end of yellowing, which allows it to carry on certain biological processes needed to convert starch to sugar and break down chlorophyll. The browning stage is also called leaf drying, where the lamina tissue is dried to a particular moisture level. Lamina color is fixed at the end of browning. The stem drying stage is referred to as the final drying process where extra moisture is removed from the stem
At the initiation of curing, the harvested tobacco is considered to be green tissue that has cell integrity, is capable of mobilizing reduced nitrogen (nitrite), and has intra-cellular compartmentalization that separates the substrates required for TSNA formation. The loss of moisture, the hydroxylation and depletion of reserve metabolites, and the continuous degradation of functional protein lead to the loss of membrane integrity, and consequently, to the loss of cell compartmentalization. Cellular degradation and moisture loss provides the opportunity for exogenous microbes to directly contact the substrates for TSNA formation. These exogenous microbes produce the NOx substrate that combines with the endogenous secondary amine alkaloids to form TSNAs during the tobacco curing process and during various types of leaf storage.
Bacterial populations on tobacco leaves are known to grow exponentially (after a “lag”) during curing as observed in traditional curing practices. Bacteria gain entrance into the tobacco leaf in large numbers through stomata or cracks formed in the leaf cuticle by tissue necrosis, particularly during lamina and stem drying of the tobacco. Bacteria also gain entrance into the tobacco leaf at any time through a damaged leaf cuticle. Damage to the leaf cuticle may occur in the field, during harvesting, during leaf transport or during curing.
The bacterial population of tobacco leaves, both primed and stalk-cut, when harvested is about 105 to 106 bacteria/gram of dry weight of tobacco leaf (Bush L. P., M. Cui, H. R. Burton, F. F. Fannin, L. Lei, and N. Dye, Recent Advances in Tobacco Science, 27, 23-46, 2001; Steel M. and W. Hempfling, Tob. Sci. Res. Conf. 54., Abst#20, 2000). The heat of the yellowing process during flue-curing and the prolonged exposure time of air-curing both result in growth of the bacterial population during yellowing. Bacterial populations may increase by 10 fold or more during this period. Once the leaf loses its membrane integrity, the nitrites react with secondary amines to form TSNA. Hence, the removal or reduction of bacterial populations in tobacco leaves or in a tobacco curing environment is desirable.
Fungi may be present on tobacco plants at harvest, during curing process and after cure. Also, some fungi produce nitrite from nitrate. Therefore, the removal or reduction of fungal growth from tobacco leaves is also desired.